Comparison of drug sensitivity among tumor cells within a tumor, between primary tumor and metastases, and between different metastases in the human tumor colony-forming assay.

The human tumor colony-forming assay was used to compare chemosensitivity among tumor cells within a primary tumor, between primary tumor and metastases, and between different metastases. No significant differences in cloning efficiency were found in any of the three comparison studies. However, considerable differences in chemosensitivities were observed between different parts of the same tumor and between the primary tumor and metastases. Two different parts of the same tumor were comparably assayed for nine primary tumors. In nine paired samples which allowed in vitro drug sensitivity testing, there was no satisfactory correlation of sensitivity to cytostatic drugs. Cell suspensions were prepared from 28 primary tumors and from metastases taken from the same patient. In 14 paired samples which formed sufficient colonies for determination of drug effect, the data showed no satisfactory correlation of chemosensitivity between a primary tumor and its metastases. Both tumor samples from different metastatic sites of the same patient formed sufficient colonies in seven of eight instances. In the seven paired samples, there was strong association of chemosensitivity (p less than 0.005). The results indicate that the reported discrepancies of in vitro and in vivo results in clinical trials using the tumor colony-forming assay for predicting resistance or sensitivity to cytostatic drugs may be due to therapeutic heterogeneity among tumor colony-forming units within a primary tumor and between a primary tumor and its metastases.

Interleukin-3 and stem cell factor modulate cell cycle regulatory factors in mast cells: negative regulation of p27Kip1 in proliferation of mast cells induced by interleukin-3 but not stem cell factor.

OBJECTIVE: Interleukin-3 (IL-3) and stem cell factor (SCF) are able to promote survival and proliferation of mast cells. However, the precise signal transduction cascades leading to mast cell proliferation are not clearly understood. Thus, we sought to define the mechanism of mast cell proliferation induced by IL-3 and SCF. MATERIALS AND METHODS: We treated murine bone marrow-derived cultured mast cells (BMCMC) with recombinant IL-3 (rIL-3) or recombinant SCF (rSCF) and examined the effects of rIL-3 and rSCF on cell cycle regulatory factors. RESULTS: Both rIL-3 and rSCF suppressed apoptosis of BMCMC. rSCF induced great proliferation of BMCMC with elevation of the proportions of cells in S and G2/M phases, whereas most BMCMC incubated with rIL-3 were arrested in the G1 phase. The G1/S phase transition is initiated by phosphorylated retinoblastoma protein (pRb), which was prominent in cells stimulated with rSCF. In contrast, rIL-3 relatively increased a dephosphorylated form of pRb in BMCMC. Compared with rIL-3, rSCF induced greater expression of cyclin-dependent kinase (CDK) 2 and CDK4, which are able to phosphorylate pRb, and cyclin D3, a partner of CDK4. BMCMC treated with rIL-3 contained a high amount of a CDK inhibitor p27Kip1 that was suppressed by pretreatment with Ro31-7549, a protein kinase C inhibitor, whereas rSCF induced weak expression of p27Kip1 in BMCMC. CONCLUSION: The results suggest that IL-3 and SCF exert their respective mitogenic effects on mast cells by modulating the expression of pRb, CDK, cyclin, and p27Kip1.

Surgical chemotherapy against lymph node metastases: an experimental study.

Accompanying surgical resection of the primary tumor is removal of its drainage lymph nodes. However, all of the minute regional lymph nodes cannot be identified and some may be left behind. If a certain anticancer agent in the form of an emulsion is injected topically into the lymph nodes, it may suppress the lymphatic metastases. Domestic rabbits were used as experimental animals, because transplantable VX2 tumors are available. The vermiform appendix was selected as the transplantation site because of its rich supply of lymph follicles, simulating lymph nodes histologically, and because the path of lymph drainage is very simple. The drainage lymph node, which is located at the root of the appendix, was selected for study. The rate of transfer of bleomycin into lymph nodes and of its sustained release from the nodes was extremely enhanced by the use of a sphere-in-oil-type emulsion--more than two times higher than in the use of a W/O emulsion. Although prolongation of survival time did not take place in animals receiving the bleomycin solution topically or intravenously, five of the seven rabbits receiving the local administration of bleomycin as a sphere-in-oil or a water-in-oil emulsion, between which differences were not found in tumor effects, survived with complete reduction of the lymph node metastases.

Antagonism of the emetic action of xylazine by alpha-adrenoceptor blocking agents.

The intramuscular injection of xylazine (2 mg/kg) evoked vomiting in 81% of the dogs studied. Adrenoceptor antagonists showing alpha 2-blocking activity, yohimbine, tolazoline and phentolamine, antagonized the xylazine-induced vomiting in a dose-dependent manner. Of these antagonists, yohimbine was the most effective, since the maximal antagonistic effect was seen at 0.5 mg/kg yohimbine, a dose at which the other drugs had less or no effect. The adrenoceptor antagonists showing alpha 1-blocking activity, prazosin and phenoxybenzamine, at the doses studied did not prevent the emesis induced by xylazine. A beta-adrenoceptor antagonists, propranolol, was ineffective in reducing xylazine-induced vomiting. The dopamine receptor antagonists, metoclopramide and domperidone, did not prevent xylazine-induced vomiting nor did yohimbine antagonize apomorphine-induced vomiting. The xylazine-induced vomiting was not prevented by atropine, naloxone or hexamethonium. These results indicate that the xylazine-induced vomiting is mediated by alpha 2-adrenoceptors and does not appear to involve beta-adrenoceptors, cholinoceptors, dopamine or opiate receptors in the emetic pathway.

Central alpha-adrenoceptor subtypes involved in the emetic pathway in cats.

The intracerebroventricular (i.c.v.) injection of clonidine, xylazine, adrenaline and methoxamine elicited dose-dependent vomiting in cats in that order of potency. The vomiting induced by clonidine, xylazine and adrenaline was antagonized by i.c.v. yohimbine and phentolamine possessing alpha 2-adrenoceptor-blocking activity, but not by prazosin showing alpha 1-adrenoceptor-blocking activity. In contrast, methoxamine-induced vomiting was antagonized by prazosin, but not by yohimbine. The vomiting induced by xylazine and adrenaline was not prevented by i.c.v. 6-hydroxydopamine treatment, but was prevented by i.c.v. reserpine treatment. Ablation of the area postrema with some damage to extremely adjacent areas abolished the vomiting induced by each alpha-adrenoceptor agonist. These results indicate that both central alpha 1- and alpha 2-adrenoceptors are involved in the emetic pathway in cats, although alpha 2-adrenoceptors seem to have the main role. It is also suggested that monoamines, and in particular 5-hydroxytryptamine in the brain, are involved in the regulation of alpha-adrenoceptor-mediated vomiting.

Effect of leukotriene C4D4 antagonist on colonic damage induced by intracolonic administration of trinitrobenzene sulfonic acid in rats.

We examined the effects of eicosanoid antagonists on colonic damage induced by trinitrobenzene sulfonic acid (TNB) in a rat inflammatory bowel model. TNB (30 mg) dissolved in 0.25 ml of 50% ethanol, was given intrarectally. The appropriate doses of ONO-1078 (a leukotriene C4D4 antagonist), ONO-4057 (a leukotriene B4 antagonist), and OKY-046 (a thromboxane A2 synthetase inhibitor) were given to obtain the same blood level, either 4 h before (pre-treatment model) or 24 h after (the post-treatment model) the administration of TNB (n = 8 in all groups). Drugs were given once daily for 6 days through a gastric feeding tube. Autopsy was performed on the 7th day. Colonic damage was assessed in terms of colonic damage scores, and myeloperoxidase (MPO) activity and eicosanoid concentrations in colonic tissues were measured. Compared with the group given TNB alone, the colonic damage score was reduced to 10% in the pre-treatment model with ONO-1078, but the score was not reduced in other groups, MPO activity was not changed in any group. The concentration of leukotriene C4 was reduced with ONO-1078 treatment, in both pre- and post-treatment models. These results demonstrated that a leukotriene C4D4 antagonist reduced colonic inflammation; however, its anti-inflammatory effect was limited in this colitis model.

Influence of Helicobacter pylori infection on development of stress-induced gastric mucosal injury.

Immediately after the Great Hanshin Earthquake in Kobe in 1995, the recurrence rate of peptic ulcer in patients infected with Helicobacter pylori was higher than that in patients in whom H. pylori had been eradicated. We evaluated the influence of H. pylori infection on stress-induced gastric mucosal injury in Mongolian gerbils and C57BL/6 mice. These animals were immersed in water for 30, 120, and 720 min 12 weeks after inoculation with H. pylori, and then killed to assess gastric mucosal damage, and to measure cytokine production (interleukin [IL]-1beta, IL-4, IL-6, and IL-10; interferon [IFN]-gamma; and tumor necrosis factor [TNF]-alpha) in the gastric tissue of the mice. The stress treatment for 30 min resulted in a significantly higher bleeding rate and bleeding index among infected gerbils and mice compared with results in uninfected animals. Conversely, the bleeding and ulcer indexes were significantly higher in uninfected gerbils after 720 min of the stress treatment than in infected gerbils. Prior to the stress treatment, gastric IL-1beta and IFN-gamma production was significantly higher in the infected group than in the uninfected group. After 120 min of the stress treatment, TNF-alpha production was increased in the infected group, and IL-1beta and IL-10 production was increased in the uninfected group. However, the production of these cytokines showed no change at 30 min of the stress treatment. These results suggest that H. pylori infection influences the development of gastric mucosal injury in the early phase of stress exposure; cytokines do not play a major role in this process.

Analysis of conjugated bile acids in bile by high-pressure liquid chromatography.

The major conjugated bile acids of man, including glycine and taurine conjugates, can be separated by high-pressure liquid chromatography (HPLC). The chromatographic column is 300 mm long with an internal diameter of 8.0 mm and is packed with Lichrosorb RP 18 (5 MICRONS). The mobile phase is methanol-water 75 : 25 (v/v) acidified to pH 2 with phosphoric acid. The eluent peaks are detected by a UV absorbance detector at a wavelength of 210 nm. The 10 conjugated bile acids are quantitatively analyzed on a single run of chromatography in less than 50 min. Deproteinization of biological samples is sufficient for the preparation of the analysis.

Platelet and brain alpha 2-adrenoceptors and cardiovascular sensitivity to agonists in dogs suffering from endotoxic shock.

We examined the changes in alpha 2-adrenoceptor binding on platelet and brain membranes of dogs treated with a non-lethal dose of endotoxin (0.1 mg/kg intravenously), and the alpha 2-adrenoceptor mediated cardiovascular effects during endotoxin shock. At 2 h, 24 h, and 7 days after endotoxin administration, the number of binding sites (Bmax) of [3H]yohimbine binding decreased and equilibrium dissociation constants (Kd) increased in platelets, whereas both Bmax and Kd decreased in either cerebral cortex or medulla oblongata. After 30 days of endotoxin administration, there were no significant differences in Bmax or Kd between the treated and untreated animals in both platelets and brain tissues. Significant positive correlations were observed for Bmax values between platelets and brain tissues, although negative correlations for Kd values between platelets and brain were not significant. Significant negative correlations were also observed between plasma catecholamine concentrations and platelet alpha 2-adrenoceptor number, and between plasma noradrenaline and medulla alpha 2-adrenoceptor number. Pretreatment with E coli endotoxin diminished cardiovascular effects such as bradycardia, hypotension, and increase in systemic vascular resistance induced by either i.v. clonidine or xylazine. This suggests that alpha 2-adrenoceptor activity is impaired in the central nervous system as well as in the peripheral vascular system during endotoxin shock. Therefore, platelets may in part represent a good model which reflects the alpha 2-adrenoceptor changes in the central nervous system and peripheral vascular system during and after endotoxin shock.

Present features of gallstones in Japan. A collective review of 2,144 cases.

A collective review of 2,144 patients operated on for cholelithiasis during the last twenty-two months has shown that younger Japanese adults have predominantly cholesterol stones in their gallbladders and that elderly persons still frequently have bilirubin stones not only in their gallbladders but also in their common bile ducts. Recent westernization of dietary habits in Japan is considered to be the most probable factor causing the increased incidence of cholesterol stones. The decreased incidence of bilirubin stones is considered to be caused by the decreased incidence of biliary infection and increased intake of proteins in food.

Cardiopulmonary effects of sevoflurane in cats: comparison with isoflurane, halothane, and enflurane.

The cardiopulmonary effects of sevoflurane (mean, 2.6, 3.8-3.9 and 5.2 per cent) were compared with those of halothane (1.2, 1.8 and 2.4 per cent), enflurane (2.4, 3.6 and 4.8 per cent) and isoflurane (1.6, 2.4 and 3.2-3.3 per cent) at end-tidal concentrations equivalent to 1, 1.5 and 2 minimal alveolar concentrations (MACs) during spontaneous or controlled ventilation (SV or CV) in 57 cats. Cats were assigned to four groups of nine animals each in SV trial and four groups of five or six animals each in CV trial. During SV, respiration rate was decreased by sevoflurane and isoflurane at 2 MAC and by enflurane at each MAC multiple when compared with control values, whereas halothane increased respiration rate at 2 MAC. The degree of hypercapnia and acidosis induced by sevoflurane was not different from that induced by isoflurane and was less than that induced by halothane at 1 to 1.5 MAC or enflurane at 2 MAC. During SV and CV, four anaesthetics decreased heart rate at 2 MAC when compared with control values, but there was no significant difference between anaesthetics. Sevoflurane, like halothane and isoflurane, induced hypotension at 2 MAC when compared with 1 MAC.

Ventricular arrhythmogenic dose of adrenaline during sevoflurane, isoflurane, and halothane anaesthesia either with or without ketamine or thiopentone in cats.

The doses of adrenaline required to induce ventricular arrhythmia during sevoflurane, isoflurane and halothane anaesthesia, either with or without infusions of ketamine (76 micrograms kg-1 min-1) or thiopentone (0.5 mg kg-1 min-1), were determined in cats. Groups of six to eight cats were maintained at end-tidal concentrations equivalent to 1.25 times the minimal alveolar concentration of each anaesthetic. The mean dose of adrenaline required to induce arrhythmia during sevoflurane anaesthesia (19.0 micrograms kg-1) was approximately 11 times higher than that required during halothane anaesthesia (1.66 micrograms kg-1) and the same as that required during isoflurane anaesthesia (19.0 micrograms kg-1). Ketamine tended to decrease the requirement of adrenaline during halothane anaesthesia, but not significantly, and did not change the requirement during isoflurane or sevoflurane anaesthesia. Thiopentone did not change the requirement for adrenaline during halothane, isoflurane or sevoflurane anaesthesia. It was concluded that either with or without ketamine or thiopentone, the effect of sevoflurane on the sensitisation of the feline myocardium to the arrhythmogenic effects of adrenaline was significantly less than that of halothane and not different from that of isoflurane.

Experimental study on lymphatic vascular changes in the development of cancer.

Sudan Black B and Sudan Blue can partly enter the lymphatics of the small intestine from the lumen with long chain fatty acids. By use of a fat emulsion saturated with them small intestinal and mesenteric lymphatics were clearly delineated. Subserosal and mesenteric lymphatics of the small intestine appeared as blue lines. With this method we studied anatomical changes of the lymphatic vessels during the development of cancer. As experimental tumor VX2 carcinoma was used. Lymphatic vessels were not be found in cancerous regions by this lymphangiographic procedure, even in the early stages of cancer. Lymphatics passing through the tumorous tissue were completely obstructed and accompanied with peripheral dilatation. Compared to the morphological changes of the blood vessels, which were studied microangiographically, the lymphatic vessels were more easily affected by the malignant growth.

Neurohormonal and metabolic effects of medetomidine compared with xylazine in beagle dogs.

This study was aimed to investigate and compare the effects of medetomidine and xylazine on the blood level of some stress-related neurohormonal and metabolic variables in clinically normal dogs, especially focusing on time and dose relations of the effects. A total of 9 beagle dogs were used for 9 groups, which were treated with physiological saline solution (control), 10, 20, 40, and 80 microg/kg medetomidine, and 1, 2, 4, and 8 mg/kg xylazine, intramuscularly. Blood samples were taken at 10 times during 24 h from a central venous catheter. Plasma norepinephrine, epinephrine, cortisol, glucose, insulin, glucagon, and nonesterified fatty acid concentrations were determined. Both medetomidine and xylazine similarly and dose-dependently inhibited norepinephrine release and lipolysis. Medetomidine suppressed epinephrine release dose-dependently with greater potency than xylazine. Xylazine also tended to decrease epinephrine levels dose-dependently. The cortisol and glucagon levels did not change significantly in any treatment group. Both drugs suppressed insulin secretion with similar potency. Both medetomidine and xylazine increased glucose levels. The hyperglycemic effect of medetomidine, in contrast with xylazine, was not dose-dependent at the tested dosages. The results suggested that the effect of medetomidine on glucose metabolism may not be due only to alpha2-adrenoceptor-mediated actions.

The antagonistic effects of atipamezole and yohimbine on stress-related neurohormonal and metabolic responses induced by medetomidine in dogs.

This study aimed to compare the antagonistic effects of atipamezole (40,120, and 320 microg/kg, IM), yohimbine (110 microg/kg, IM), and saline on neurohormonal and metabolic responses induced by medetomidine (20 microg/kg, IM). Five beagle dogs were used in each of the 5 experimental groups in randomized order. Blood samples were taken for 6 h. Medetomidine significantly decreased norepinephrine, epinephrine, insulin, and nonesterified fatty acid levels, and increased plasma glucose levels. Both atipamezole and yohimbine antagonized these effects. The reversal effect of atipamezole was dose-dependency, except on epinephrine. Yohimbine caused prolonged increases in plasma norepinephrine and insulin levels compared to atipamezole, possibly because of its longer half-life elimination. Only yohimbine increased the cortisol levels. Neither glucagon nor lactate levels changed significantly. Based on these findings, when medetomidine-induced sedation is antagonized in dogs, we recommend using atipamezole IM, from 2- to 6-fold the dose of medetomidine, unless otherwise indicated.

Antagonistic effects of alpha-adrenoceptor blocking agents on reticuloruminal hypomotility induced by xylazine in cattle.

The intravenous injection of a standard dose (0.05 mg/kg) of xylazine inhibited reticuloruminal motility in cattle. Pretreatment with adrenoceptor antagonists showing alpha 2-blocking activity, tolazoline (0.5 mg/kg) and yohimbine (0.2 mg/kg), antagonized the xylazine-induced reticuloruminal amotility. Tolazoline was more effective than yohimbine, since an antagonistic effect was not seen at 0.5 mg/kg yohimbine, and yohimbine at 0.2 mg/kg was less effective than tolazoline at 0.5 mg/kg. An adrenoceptor antagonist showing alpha 1-blocking activity, prazosin, did not prevent the inhibition of reticuloruminal motility by xylazine. The xylazine-induced reticuloruminal amotility was also not prevented by either a dopamine receptor antagonist, domperidone, or an opiate receptor antagonist, naloxone. These results suggest that xylazine inhibits bovine reticuloruminal motility through its activation of alpha 2-adrenoceptors, and show that tolazoline can be used as a specific antagonist of xylazine in studies of the alpha-adrenergic influence on reticuloruminal motility in cattle.

Anesthetic potency and cardiopulmonary effects of sevoflurane in goats: comparison with isoflurane and halothane.

The anesthetic potency and cardiopulmonary effects of sevoflurane were compared with those of isoflurane and halothane in goats. The (mean +/- SD) minimal alveolar concentration (MAC) was 0.96 +/- 0.12% for halothane, 1.29 +/- 0.11% for isoflurane, and 2.33 +/- 0.15% for sevoflurane. Cardiopulmonary effects of sevoflurane, halothane and isoflurane were examined at end-tidal concentrations equivalent to 1, 1.5 and 2 MAC during either spontaneous or controlled ventilation (SV or CV). During SV, there were no significant differences in respiration rate, tidal volume and minute ventilation between anesthetics. Dose-dependent decreases in both tidal volume and minute ventilation induced by halothane were greater than those by either sevoflurane or isoflurane. Hypercapnia and acidosis induced by sevoflurane were not significantly different from those by either isoflurane or halothane at 1 and 1.5 MAC, but were less than those by halothane at 2 MAC. There was no significant difference in heart rate between anesthetics during SV and CV. During SV, all anesthetics induced dose-dependent decreases in arterial pressure, rate pressure product, systemic vascular resistance, left ventricular minute work index and left ventricular stroke work index. Systemic vascular resistance with isoflurane at 2 MAC was lower than that with sevoflurane. During CV, sevoflurane induced dose-dependent circulatory depression (decreases in arterial pressure, cardiac index, rate pressure product, systemic vascular resistance, left ventricular minute work index and right ventricular minute work index), similar to isoflurane. Halothane did not significantly alter systemic vascular resistance from 1 to 2 MAC.

Cliniconeuropathologic findings of familial frontal lobe epilepsy in Shetland sheepdogs.

We examined an epileptic focus by electroencephalography (EEG) by using an international 10-20 electrode system in 11 Shetland sheep dogs affected with familial idiopathic epilepsy. We also performed an evaluation of the amino acids in the cerebrospinal fluid (CSF) and a pathologic examination of the brains of 8 dogs that died from status epilepticus. Continuous electroencephalography demonstrated that an epileptic focus was initially detected in the frontal lobe, particularly the internal area, and that paroxysmal foci developed diffusely in other lobes of affected dogs with recurrent convulsions. The EEG analyses indicated spike and sharp wave complexes, which were considered to be paroxysmal discharges. An increased value for glutamate or aspartate was found in the CSF of some epileptic dogs. Histologically, acute neuronal necrosis and astrocytosis were distributed predominantly in the cingulate cortex and internal area of frontal cortex, less frequently in other areas of the cerebrum. The results of this study suggest that, initially, the dogs have an epileptic focus in the frontal lobe, and that the focus extends gradually to other areas of the cerebrum. Based on the distribution of neuronal necrosis and astrocytosis, acute neuronal damage may be related to the superexcitation of neurons following epilepsy.

Sevoflurane anesthesia following administration of atropine-guaifenesin-thiopental in spontaneous-breathing adult cattle.

The effect of sevoflurane-oxygen anesthesia at surgical depth on clinically important features was evaluated in spontaneously breathing cattle given atropine-guaifenesin-thiopental. The mean end-tidal sevoflurane concentrations ranged from 1.4 to 2.0% for 75 min maintenance. Recovery from anesthesia was extremely rapid and smooth. Heart rate increased and arterial pressure was higher during anesthesia, but respiratory rate did not change significantly. Arrhythmia was not observed. Minute ventilation was low due to decreased tidal volume during anesthesia, and changes in blood gases and pH showed mild respiratory acidosis. There were no marked changes in hematologic or serum biochemical values for 7 days postanesthesia. These findings suggest that sevoflurane is an effective inhalant anesthetic showing rapid recovery from anesthesia.

Differentiation of stromal-vascular cells isolated from canine adipose tissues in primary culture.

A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 microM indomethacin, 10 microg/ml insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental). demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 +/- 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development.